4. Discussion
Although a bioreactor with reciprocal mixing cannot be simply compared to conventional bioreactors with rotary paddles, the cultivation conditions of CHO cells in both bioreactors, in terms of cell growth, damage, and lactate production were compared in this report. There was no significant difference between the bioreactors in terms of cell proliferation and density during exponential phase. However, in the reciprocal bioreactor, the transition from stationary to death phase was slower, and high cell viability was maintained for a longer period than in the bioreactor with rotary paddles. Moreover, lactate dehydrogenase (LDH) activity in the medium of reciprocal bioreactor was much lower than in the rotary bioreactor. Since LDH is a cell damage marker, shear stress on cells in the reciprocal bioreactor was shown to be overall lower than in the rotary bioreactor. The condition of cells, after entering early stationary phase, is important for industrial production of targeted proteins. Therefore, maintaining high cell viability after stationary phase is critical for high-quality production.
When shear stress in bioreactors was analyzed by CFD, strong shear stress generated by separation vortex was observed around the rotary paddles, and cells were found to be exposed to it throughout the cultivation process. On the other hand, maximum shear stress in reciprocal mixing was observed around the plates when they turned at the top and bottom dead points. Shear stress in reciprocal mixing was minimized when the plates passed through the middle point of stroke. This non-steady-state mixing of reciprocal motion could dramatically reduce the accumulation of cell damage, thereby affecting the physiological conditions of living cells.
Influence of shear stress on cell damage has been investigated not only in bioreactors, but also in microfluid devices and blood vessels (Mitchell, 2013; Odeleye, 2014; Model, 2014). Although many researchers have tried to clarify how shear stress affects gene expression and physiological conditions (Nurhayati, 2018; Jayagopal, 2019), the kind of cellular systems that sense the effect of shear stress on gene expression still remains unknown. Even if a type of shear stress induced gene expression in one cell type, it may not affect the same in other cell lines (Akimoto, 2000; Novak, 2019). Therefore, how reciprocal mixing could influence gene expression and intracellular physiological conditions, resulting in the maintenance of high cell viability and target protein expression, needs to be clarified in future.
Recently, Eto’s group reported a bioreactor with reciprocal mixing to be effective for platelet generation from human iPS-derived megakaryocytes (Ito, 2018). Platelet biogenesis from megakaryocytes requires blood flow-dependent shear stress (Junt, 2008). While other bioreactors with rotary paddles or culture bags could not produce sufficient and appropriate levels of shear stress, a bioreactor with reciprocal motion could generate proper turbulent energy in the culture medium, thereby inducing in-vitro thrombopoiesis.
Development of a bioreactor, till date, has mainly focused on the growth of cultured organism, production of targeted compounds or proteins, and homogeneity of culture broth. A wide variety of ideas, including different kinds of mixing impellers, and rotating or see-sawing culture vessels have been developed over the years (Birch, 1990; Rotenberg, 2012). However, mixing and dispersing actions are inversely proportional to shear stress, in case of conventional bioreactors, making it difficult to optimize the culture conditions (Wyma, 2018). In this study, we have introduced a novel concept of bioreactors for culturing animal cells, and clarified the specific characteristics of a bioreactor with reciprocal mixing in comparison to those of a conventional bioreactor with rotary paddles. Although the reason behind the appreciable effect of reciprocal mixing on cell growth remains unknown, shear stress generated by reciprocal motion might be physiologically acceptable for cell growth. Studies are ongoing for understanding the processes occurring in cells when they are exposed to reciprocal shear stress.